Paul K. Herman

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.

Stationary phase in yeast

Eukaryotic cell proliferation is controlled by specific growth factors and the availability of essential nutrients. If either of these signals is lacking, cells may enter into a specialized nondividing resting state, known as stationary phase or G(0). The entry into such resting states is typically accompanied by a dramatic decrease in the overall growth rate and an increased resistance to a variety of environmental stresses. Since most cells spend most of their life in these quiescent states, it is important that we develop a full understanding of the biology of the stationary phase/G(0) cell. This knowledge would provide important insights into the control of two of the most fundamental aspects of eukaryotic cell biology: cell proliferation and long-term cell survival. This review will discuss some recent advances in our understanding of the stationary phase of growth in the budding yeast, Saccharomyces cerevisiae.

The cAMP-dependent protein kinase signaling pathway is a key regulator of P body foci formation.

In response to stress, eukaryotic cells accumulate mRNAs and proteins at discrete sites, or foci, in the cytoplasm. However, the mechanisms regulating foci formation, and the biological function of the larger ribonucleoprotein (RNP) assemblies, remain poorly understood. Here, we show that the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae is a key regulator of the assembly of processing bodies (P bodies), an RNP complex implicated in mRNA processing and translation. The data suggest that PKA specifically inhibits the formation of the larger P body aggregates by directly phosphorylating Pat1, a conserved constituent of these foci that functions as a scaffold during the assembly process. Finally, we present evidence indicating that P body foci are required for the long-term survival of stationary phase cells. This work therefore highlights the general relevance of RNP foci in quiescent cells, and provides a framework for the study of the many RNP assemblies that form in eukaryotic cells.

Autophosphorylation Within the Atg1 Activation Loop Is Required for Both Kinase Activity and the Induction of Autophagy in Saccharomyces cerevisiae

Autophagy is an evolutionarily conserved degradative pathway that has been implicated in a number of physiological events important for human health. This process was originally identified as a response to nutrient deprivation and is thought to serve in a recycling capacity during periods of nutritional stress. Autophagy activity appears to be highly regulated and multiple signaling pathways are known to target a complex of proteins that contains the Atg1 protein kinase. The data here extend these observations and identify a particular phosphorylation event on Atg1 as a potential control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1 orthologs. Replacing this threonine with a nonphosphorylatable residue resulted in a loss of Atg1 protein kinase activity and a failure to induce autophagy. This phosphorylation required the presence of a functional Atg1 kinase domain and two known regulators of Atg1 activity, Atg13 and Atg17. Interestingly, the levels of this modification were found to increase dramatically upon exposure to conditions that induce autophagy. In addition, T226 phosphorylation was associated with an autophosphorylated form of Atg1 that was found specifically in cells undergoing the autophagy process. In all, these data suggest that autophosphorylation within the Atg1 activation loop may represent a point of regulatory control for this degradative process.

Processing Body and Stress Granule Assembly Occur by Independent and Differentially Regulated Pathways in Saccharomyces cerevisiae

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.

Using Substrate-Binding Variants of the cAMP-Dependent Protein Kinase to Identify Novel Targets and a Kinase Domain Important for Substrate Interactions in Saccharomyces cerevisiae

Protein kinases mediate much of the signal transduction in eukaryotic cells and defects in kinase function are associated with a variety of human diseases. To understand and correct these defects, we will need to identify the physiologically relevant substrates of these enzymes. The work presented here describes a novel approach to this identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. This approach takes advantage of two catalytically inactive PKA variants, Tpk1K336A/H338A and Tpk1R324A, that exhibit a stable binding to their substrates. Most protein kinases, including the wild-type PKA, associate with substrates with a relatively low affinity. The binding observed here was specific to substrates and was dependent upon PKA residues known to be important for interactions with peptide substrates. The general utility of this approach was demonstrated by the ability to identify both previously described and novel PKA substrates in S. cerevisiae. Interestingly, the positions of the residues altered in these variants implicated a particular region within the PKA kinase domain, corresponding to subdomain XI, in the binding and/or release of protein substrates. Moreover, the high conservation of the residues altered and, in particular, the invariant nature of the R324 position suggest that this approach might be generally applicable to other protein kinases.

Antagonistic Interactions Between the cAMP-Dependent Protein Kinase and Tor Signaling Pathways Modulate Cell Growth in Saccharomyces cerevisiae

Eukaryotic cells integrate information from multiple sources to respond appropriately to changes in the environment. Here, we examined the relationship between two signaling pathways in Saccharomyces cerevisiae that are essential for the coordination of cell growth with nutrient availability. These pathways involve the cAMP-dependent protein kinase (PKA) and Tor proteins, respectively. Although these pathways control a similar set of processes important for growth, it was not clear how their activities were integrated in vivo. The experiments here examined this coordination and, in particular, tested whether the PKA pathway was primarily a downstream effector of the TORC1 signaling complex. Using a number of reporters for the PKA pathway, we found that the inhibition of TORC1 did not result in diminished PKA signaling activity. To the contrary, decreased TORC1 signaling was generally associated with elevated levels of PKA activity. Similarly, TORC1 activity appeared to increase in response to lower levels of PKA signaling. Consistent with these observations, we found that diminished PKA signaling partially suppressed the growth defects associated with decreased TORC1 activity. In all, these data suggested that the PKA and TORC1 pathways were functioning in parallel to promote cell growth and that each pathway might restrain, either directly or indirectly, the activity of the other. The potential significance of this antagonism for the regulation of cell growth and overall fitness is discussed.

An Atg13 Protein-mediated Self-association of the Atg1 Protein Kinase Is Important for the Induction of Autophagy

Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.

A new method of measuring dimensional change.

A new method of measuring linear dimensional change in denture base resins is presented and its precision in determining distances between two points was investigated. Chrome steel ball bearings 1.57 mm in diameter were invested in a stone mold and were used to record measurements by a reflection from the surface of the ball bearings. The accuracy of this method was determined by performing repeated measurements between two points in a stone mold. Random measurements were made to an accuracy of 0.002 mm. The dimensional change of light-cured, heat-cured, and self-cured denture base resins was also determined with this method, followed by an analysis of variance of the data. The findings support the accuracy and reproducibility of this new method in recording linear measurements. The dimensional changes of all three resins were different from each other after the specimens were processed and stored in distilled water for 7 days at 37 degrees C.

The regulation of autophagy in eukaryotic cells: do all roads pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be refractory to cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signalingpathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.